Scientific abstract Infiltrating lymphocytes, macrophages and astrocytes are major drivers of multiple sclerosis (MS) lesion pathology, yet their exact roles in lesion formation are still poorly understood. Key to the function of these cells is their phenotypic heterogeneity as well as their spatial interactions within the lesion environment. This information cannot be obtained with standard immunohistochemistry, which does not allow for high parameter profiling, or with single cell approaches, which do not capture spatial information. We propose here to use a novel approach, highly multiplexed imaging, to profile single cells in acute and chronic active MS lesions. To this end, we will employ iterative indirect immunofluorescence imaging (4i), an immunofluorescence-based method which allows for staining with >40 antibodies on a single histological section, and subsequently perform spatial analysis using a powerful computational pipeline. In aim 1, we will use an antibody panel against 32 markers of myeloid cell and astrocyte function to (i) characterize macrophage and astrocyte subpopulations, (ii) determine their distribution within MS lesions and (iii) define their interactions with other cell types such as lymphocytes. Thus, our analysis will provide phenotypes and specific phenotypic interactions that drive demyelination in acute lesions, and low-grade inflammation in chronic active lesions, In aim 2, we will comprehensively assess immune checkpoints in MS lesions, a highly complex and redundant system, that modulates T cell activation. We will use a total of 41 antibodies to determine expression of co-inhibitory/stimulatory receptors on different T cell subpopulations, and their corresponding ligands on surrounding cells. This will deliver the specific checkpoints that drive loss of tolerance in lymphocytes in acute and chronic MS lesions. By profiling complex cellular states within spatial context in MS lesions, we will obtain previously inaccessible histological information. Our data will ultimately provide targets for therapeutic intervention during acute lesion formation and in chronic progressive MS.